The innate immune system utilizes germline-encoded pattern recognition receptors, such as Toll like receptors (TLRs), to sense molecular patterns that are common in microbial pathogens but rare or non-existent in the host. TLR9 is required for antiviral responses. Although TLR9 has been implicated in the recognition of viral DNA, it is not clear how this receptor discriminates between viral and host DNA. Until recently, TLR9 was thought to exclusively recognize unmethylated CpG motifs in DNA, a characteristic frequent in viral DNA but rare in cellular DNA. However, this model, drawn mostly from studies using DNA with modified backbone, has recently been challenged by a report indicating that natural (unmodified) DNA stimulates TLR9 regardless of the presence of CpG motifs. Moreover, recent work from the laboratory of our collaborator shows that TLR9 is enzymatically processed after synthesis to generate a truncated form that binds DNA in the endosome and lysosome. Preliminary data indicate that the full-length TLR9 (used in all the previous studies aimed at characterizing molecular interactions between TLR9 and DNA) and the physiological cleaved form of TLR9 might have distinct affinities for ODNs. Thus, the sequences that are recognized as TLR9, and in particular the physiological (truncated) form in natural DNA, remain to be determined. In this proposal, we aim to identify all the sequences (up to 8mers, over 65,000 possibilities) that are best recognized by the cleaved form of TLR9 using microfluidic technology. We will then determine which sequences stimulate or repress TLR9 activity. Finally, we will examine whether stimulatory sequences are statistically underrepresented in the genome of DNA viruses, as suggested by our preliminary data. Importantly, the identification of highly stimulatory and inhibitory motifs will be key information for the design and/or improvement of DNA-based vaccines as well as gene therapy vectors. PUBLIC HEALTH RELEVANCE: TLR9 has been implicated in the recognition of different families of viruses including herpesviruses, poxviruses and adenoviruses. This receptor is activated in the endosomes and lysosomes upon interaction with DNA. At the molecular level, TLR9 is thought to recognize nucleic acid modifications and nucleic acid sequences (in particular unmethylated CpG motifs), which are enriched in viruses but suppressed in host genomes. However, two recent reports have challenged this model. One suggests that TLR9-DNA interaction is largely sequence-independent. A second paper showed that the TLR9 form that interacts with DNA is not full length (as previously thought) but a truncated form lacking a large portion of its extracytoplasmic domain. Here, we propose to identify all sequence motifs (up to 8mers) that stimulate or inhibit the physiological (truncated) form of TLR9 and determine whether the frequency of these sequences are modulated in DNA viruses.